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HomeNucleic Acid Labeling & DetectionRandom Primed DNA Labeling Kit Protocol Troubleshooting

Random Primed DNA Labeling Kit Protocol Troubleshooting

The Random Primed DNA Labeling Kit (Product No. 11004760001) is for the radioactive and nonradioactive labeling of DNA with modified deoxyribonucleoside triphosphate using random oligonucleotides as primers. In this method, the complementary DNA strand of denatured DNA is synthesized by Klenow polymerase using the 3′-OH termini of the random oligonucleotides as primers.

Random Primed DNA Labeling Kit Protocol Troubleshooting

DNA-binding Proteins

  • Random Primed Labeling probes are not recommended for detection of DNA-binding proteins via gel shift:
  • DIG-labeled DNA probes produced by random primed labeling yield DNA with incorporated DIG-dUTP every 20-25th nucleotide. This can interfere with protein-DNA binding when the recognition sequence is altered. For best results, use DNA probes generated by 3 ' end labeling (e.g., DIG-labeled oligonucleotides) for these applications.

Background in Southern Blots

  • High background can be due to inefficient labeling. Make sure kit reagents are not degraded. Klenow polymerase is heat labile and should be stored at -20 °C. Keep Klenow on ice or in a portable cold unit.
  • DNA template purity is important for the amount of labeled DNA generated. Linear template DNA preparations should be as free of contaminants as possible: dNTPs in the triphosphate mixture are susceptible to dephosphorylation by kinase activity; random primers are susceptible to nucleases; and Klenow polymerase can be inhibited by a variety of protein contaminants.
  • Complete DNA template denaturation is critical. Heat the template to 95 °C in a dilute buffer for at least 5 minutes. Add the remaining reagents immediately to avoid renaturation during cooling of the template.
  • Reduced labeling efficiency due to template contamination can sometimes be overcome by adding additional Klenow polymerase (increase to 2-4 μL, 20-40 units), and/or increasing the incubation time.
Materials
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